Glutamine

Overexpression and editing experiments were carried out using the FLO-1 esophageal adenocarcinoma cell line obtained from the ECACC General Cell Collection and CP-A (KR-42421) Barrett’s Esophagus cells from ATCC (catalogue number CRL-4027). FLO-1 cells were grown at 37 °C and five per cent CO₂ in DMEM + 2 mM Glutamine + 10% FBS (Biosera) + 1/10,000 units of penicillin–streptomycin. CP-A cells were grown at 37 °C and five per cent CO₂ in Keratinocyte serum-free medium with 50 µg ml⁻¹ bovine pituitary extract and 5 ng ml⁻¹ recombinant human EGF (17005042, Thermo Fisher). For passaging of CP-A cells, 250 mg L⁻¹ soybean trypsin inhibitor in PBS was used (17075029, Thermo Fisher). Gene knockdown experiments were performed on OE19 cells obtained from the Francis Crick Institute cell services, OE33 cells obtained from the ECACC General Cell Collection and MFD1 cells obtained from the OCCAMS Consortium. OE19 and OE33 cells were grown in RPMI + 2 mM Glutamine + 10% FBS (Biosera) + 1/10,000 units of penicillin–streptomycin. MFD1 cells were grown in DMEM + 2 mM Glutamine + 10% FBS (Biosera) + 1/10,000 units of penicillin–streptomycin. All cells were maintained at 37 °C and five per cent CO₂, validated by short tandem repeat analysis and routinely checked for mycoplasma contamination.

The effect of fresh, 1- and 28-d leachates on the metabolic activity of two cell types was assessed using the 3-(4,5 dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay²¹ . ATCC CCL-92 mouse 3T3 cells and primary human dental pulp cells (HDPCs) harvested from healthy root canals²² of two intact premolars of a 15-year old patient were used. Ethical approval was obtained from the Birmingham Community Healthcare NHS Foundation Trust (REC Ref.: 14/EM/1128, issued on 05 June 2018).
Cells were cultured in 75 cm² flasks with 15 ml Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, supplemented with 1% penicillin/streptomycin, 2 mM glutamine with 10% heat-inactivated foetal bovine serum (Biosera, East Sussex, UK) (DMEM-10FBS) at 37 °C with 5% CO₂ in a humidified atmosphere. Cells were subcultured at 70–80% confluency and seeded in 96-well plates at 10,000 for 3T3 cells and 6,000 for HDPCs in 100 μl DMEM-10FBS and incubated for 24 h. Passages 2–7 were used.

Human White colon adenocarcinoma (Caco-2) cells (European Collection of Cell Cultures (ECACC, UK) were isolated from a primary colonic tumor in a 72-year-old White male using the explant culture technique. During routine subculture, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with the supplementation of 10% fetal bovine serum (FBS), 1% non-essential amino acid and penicillin–streptomycin solution, at 37 °C, in an incubator containing 5% CO₂. The passage number of Caco-2 cells was 25–40 in the study.
HUVEC/TERT 2 was obtained from ATCC (ATCC, Manassas, VA, USA). The cell line was isolated from the vascular endothelium of a White female patient. Cells were maintained in M199 with the supplementation of 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% amphotericin B, 2 mM glutamine (Biosera, Nuaille, France), and Endothelial Cell Growth Medium-2 (Lonza, Basel, Switzerland), at 37 °C, in a Galaxy 170R incubator under 5% CO2 (Eppendorf, Hamburg, Germany). Adhesion of the cells was support with a 0.1% gelatin solution.